Human pluripotent stem (hPS) cells have the ability to self-renew indefinitely and to differentiate into many different cell types. They therefore potentially offer an unlimited supply of a variety of human cell types for developmental research, disease modeling, drug screening, environmental chemical testing, predictive toxicology and ultimately cell replacement therapies to treat many currently intractable degenerative diseases. The source of hPS cells is no longer limited to human embryonic stem (hES) cells. Reprogramming technology has advanced the field by enabling the routine production of hPS cells, known as induced pluripotent (iPS) cells, from easily obtained normal and diseased donor cells such as skin or hair. As a result, the number and genetic diversity of hPS cell lines available for research and development has greatly increased in recent years. This trend is likely to continue with government and private initiatives to bank thousands of iPS cell lines. However, our ability to characterize and validate hPS cells has not kept pace with advances in the rate of production. Variation in iPS cell line quality and differentiation propensity is a critcal issue that requires careful characterization of at least 6 clones for each iPS cell line. Current methods of verifying pluripotency and characterizing differentiation involve laborious and time consuming immunocytochemical (ICC) analysis of iPS clones for multiple markers of in vitro differentiation for each of 3 primary germ layers. In vivo testing in SCID mice for teratoma formation takes 6-12 weeks and is therefore not practical for initial iPS screening. Thus there is a critical unmet need for more efficient in vitro iPS screening methods to assess quality and to determine in vitro differentiation propensity. Here we propose to develop a peptide targeted quantum dot (PTQD) assay for differentiation to each germ layer. We have designed the PTQD assay to be simpler, faster, more sensitive and more convenient than ICC. The assay uses PTQDs for live cell targeting and detection in a single step, making the assay more amenable to multiplexing and automation than ICC. Importantly, the assay preserves the cells live for further use. In phase I, we will develop a multiplexed PTQD assay for definitive endoderm (DE) and identify additional peptides for ectoderm and mesoderm assays. Having demonstrated the feasibility of a multiplexed PTQD assay for DE, we will in phase II, develop ectoderm and mesoderm PTQD assays using peptides from phase I. It is very useful for stem cell researchers to know the propensity of each iPS cell line to differentiate in vitro toward one germ line or another so that they can choose an appropriate line to fit their individual application. Having developed probes for each germ line, we aim by the end of phase II to develop a simple differentiation propensity assay using color coded PTQDs that results in a different color readout for each of the 3 germ layers.